The MMTV LTR adopts a specific nucleoprotein organization when introduced into cells. This structure involves the positioning of six nucleosome families (A-F) over the 1300 base pair LTR. A specific chromatin transition is induced by the binding of steroid receptors to the B nucleosome. Our previous work showed that each phased nucleosome corresponds to a family of octamer cores positioned in that region. Thus, the low resolution phasing pattern results from the frequency-biased occupancy of a subset of these frames. Using a new assay, the linked restriction enzyme assay, we showed that the chromatin alteration does not correspond to a single nucleosome event, but involves both the B and C nucleosome families. We have now developed in vitro assembly systems that support the reconstitution of MMTV promoter DNA into ordered nucleosome arrays. Our results indicate that the addition of purified glucocorticoid receptor to the assembled arrays induces a chromatin transition that is directly analogous to that observed in vivo. Using this system, we discovered a new set of hormone response elements on the C nucleosome family, and demonstrated that the location of the cis-acting response elements determine the in vivo boundaries of the hypersensitive transition. Using the in vitro assembly system, we showed that GR-dependent enzyme access to remodeled chromatin only occurs in the presence of ATP and a HeLa nuclear extract. In the absence of ATP and a source of remodeling activity, GR is statically bound to chromatin. These findings are consistent with observations in living cells indicating that GR acts at the MMTV promoter in hit-and-run mode, and refute current dogma which argues that steroid receptors are statically bound to hormone response elements in the presence of ligand.